Binding of porphyrin to human serum albumin. Structure-activity relationships.

The equilibrium binding of hydroxyethyl vinyl deuteroporphyrin (HVD) and of irreversible porphyrin aggregates to human serum albumin was studied at the molecular level. This protein may function as an endogenous drug carrier for porphyrins in photodynamic therapy of tumours. HVD-protein binding studies revealed two types of binding sites, which are attributed to the two HVD isomers. The binding constant for the high-affinity isomer, 2.1 (+/- 0.3) x 10(8) M-1, is similar to that previously determined for protoporphyrin. At the same time the binding constant for the lower-affinity HVD isomer, 1.8(+/- 0.3) x 10(6) M-1, is similar to that previously determined for haematoporphyrin. Irreversible porphyrin aggregates were purified from the haematoporphyrin derivative and from Photofrin and are defined by spectral and chromatographic data. Gel-exclusion studies indicate that the dominant size of these aggregates is ten porphyrin monomeric units. The protein-binding constant of these aggregates is 1.7(+/- 0.2) x 10(5) M-1, with four binding sites per protein molecule. The distinction between the HVD isomers along the porphyrin-protein affinity sequence gives insight into the relationship of porphyrin structure to porphyrin-albumin binding. On the basis of this study an evaluation of human serum albumin as an endogenous carrier for porphyrins (at various aggregation states) in photodynamic therapy of tumours is presented.     

The crystal structure of halofantrine-ferriprotoporphyrin IX and the mechanism of action of arylmethanol antimalarials

The crystal structure of the complex formed between the antimalarial drug halofantrine and ferriprotoporphyrin IX (Fe(III)PPIX) has been determined by single crystal X-ray diffraction. The structure shows that halofantrine coordinates to the Fe(III) center through its alcohol functionality in addition to π-stacking of the phenanthrene ring over the porphyrin. The length of the Fe(III)-O bond is consistent with an alkoxide and not an alcohol coordinating group. The iron porphyrin is five coordinate and monomeric. Changes in the electronic spectrum of Fe(III)PPIX upon addition of halofantrine base in acetonitrile solution are almost identical to those observed upon addition of quinidine free base in the same solvent. This suggests homologous binding. Molecular mechanics modeling of Fe(III)PPIX complexes of quinidine, quinine, 9-epiquinine and 9-epiquinidine based on this homology suggests that the antimalarially active quinidine and quinine can readily adopt conformations that permit formation of an intramolecular salt bridge between the protonated quinuclidine tertiary amino group and unprotonated heme propionate group, while the inactive epimers 9-epiquinidine and 9-epiquinine have to adopt high energy conformations in order to accommodate such salt bridge formation. We propose that salt bridge formation may interrupt formation of the hemozoin precursor dimer formed during the heme detoxification pathway and so account for the strong activity of the two active isomers.     

A probable case of prehistoric kidney stone disease from the northern Cape Province, South Africa

During the excavation of isolated graves along the north shore of the Orange River in the Cape Province of South Africa, a skeleton was uncovered that showed two large calcareous deposits near the lumbar region of the vertebral column. The individual was an adult female of about 55 years of age at the time of death. The calcified residue did not in any way resemble the external or internal anatomical form of a kidney, but X-ray powder diffraction analysis and scanning electron microscopy identified the material as apatite (Ca10(PO4)6 (OH)2), a common constituent of human urinary calculi. It is postulated that the bilateral calcification was the result of a chronic renal disorder. Although it is rarely possible to identify the cause of death from skeletal remains alone, the disorder as seen in this specimen would appear to have been very severe and may well have been the ultimate cause of death.     

Effect of Processing Route on the Surface Properties of Amorphous Indomethacin Measured by Inverse Gas Chromatography

The aim of this study was to investigate the effect of processing route (i.e., quench cooling and ball milling) on the surface energy heterogeneity and surface chemistry of indomethacin (IMC). Recently developed inverse gas chromatography (IGC) methodology at finite concentrations was employed to determine the surface energy distributions of crystalline, quench cooled and milled IMC samples. Surface properties of crystalline and processed IMC were measurably different as determined by the IGC and other conventional characterization techniques: differential scanning calorimetry and powder X-ray diffraction. Quench cooled IMC was in fully amorphous form. Milled IMC showed no amorphous character by calorimetric or X-ray diffraction studies. It was demonstrated that both processed IMC samples were energetically more active than the crystalline IMC. In particular, milled IMC exhibited a relatively higher dispersive surface energy and higher surface basicity (electron donor capability). This may be attributed to the creation of surface defect sites or exposure of higher energy crystal facets during the milling process. This study confirms that processing route has notable influence on the surface energy distribution and surface acid–base character. IGC was demonstrated as a powerful technique for investigating surface properties of real-world, heterogeneous pharmaceutical materials.Key words: heterogeneity, indomethacin, inverse gas chromatography, surface disorder, surface energy     

High-performance liquid chromatographic analysis of porphyrins and their isomers with radial compression columns

Porphyrin methyl esters and the isomers of uroporphyrin and heptacarboxylic porphyrin were separated by high-performance liquid chromatography. Isocoproporphyrin was also separated from coproporphyrin. By slight modifications to the solvent mixture, the separation of all biological polycarboxylic porphyrins was achieved. These separations were made possible through the high efficiency of 10- or 5-μm particle-size Radial-PAK cartridges, which have been used in the separation of porphyrins in various excreta and tissues in a number of porphyrias.     

The effects of lipoxin A and lipoxin B on functional responses of human granulocytes

Lipoxin A and lipoxin B (LXA and LXB) are formed from arachidonic acid by leukocyte 5- and 15-lipoxygenases. We have assessed the effects of synthetic lipoxins on functional responses of human granulocytes. LXA stimulated migration at 1 nM. The effect was highly stereospecific, since e.g. 6S-LXA and LXB were less active than LXA. Neither synthetic LXA nor several of its stereoisomers provoked degranulation or aggregation. LXB and its isomers did not induce any of these functional responses. These results indicate that migratory granulocyte responses to LXA are highly stereospecific.     

Physical Studies on Melanins: II. X-Ray Diffraction

A number of purified natural and synthetic melanins have been examined by X-ray diffraction. A consistent finding with all samples was the lack of structure in the diffraction pattern corresponding to any significant crystallinity in these melanin preparations. A diffuse ring, centered at a Bragg spacing of 3.4 A was consistently found in samples of melanin from animal sources, and a similar ring at 4.2 A in all melanins obtained from plants. Models for these two polymer types, based upon the current concept that they primarily involve indole and catechol monomeric units respectively, were then evaluated by a Monte Carlo method. From the comparison of the observed spacings with the calculated ones it was concluded that the 4.2 A spacing in the catechol melanins is probably related to the average interaction between adjacent monomeric units, with mutually random orientations. The 3.4 A spacing observed in indole melanins appears to derive from the tendency of indole monomers (probably of adjacent chains) tending to aggregate in near parallel stacks. Some randomness in the form of translations and rotations parallel to the planar groups is consistent with the diffraction patterns. An interesting finding was that the diffraction pattern of synthetic melanin prepared by the alkaline auto-oxidation of catechol gave the 3.4 A spacing found in the indole melanins of natural origin.     

Determination of cellulose crystallinity from powder diffraction diagrams

One‐dimensional (1D) (spherically averaged) powder diffraction diagrams are commonly used to determine the degree of cellulose crystallinity in biomass samples. Here, it is shown using molecular modeling how disorder in cellulose fibrils can lead to considerable uncertainty in conclusions drawn concerning crystallinity based on 1D powder diffraction data alone. For example, cellulose microfibrils that contain both crystalline and noncrystalline segments can lead to powder diffraction diagrams lacking identifiable peaks, while microfibrils without any crystalline segments can lead to such peaks. This leads to false positives, that is, assigning disordered cellulose as crystalline, and false negatives, that is, categorizing fibrils with crystalline segments as amorphous. The reliable determination of the fraction of crystallinity in any given biomass sample will require a more sophisticated approach combining detailed experiment and simulation. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 67–73, 2015.     

Effect of creatine on contents of myosin heavy chain and myosin-heavy-chain mRNA in steady-state chicken muscle-cell cultures.

The i.r. spectra of bilirubin isomers that differ in number and position of vinyl groups were examined to verify the assignment of the 988 cm-1 peak of bilirubin (991 cm-1 peak in calcium bilirubinate) to its pendant vinyl groups. There were only small changes in this peak with changes in position of vinyl groups (exo-2- and -18-vinyl versus endo-3- and -17-vinyl), but progressive loss of peaks in this region was observed when vinyl groups were reduced to ethyl groups (dihydrobilirubin and mesobilirubin). Methylvinylmaleimide, a monopyrrole derived from the outer (A and D) rings of bilirubin, has a pendant vinyl group and exhibits a prominent peak at 986 cm-1, but haematinic acid methyl ester derived from the inner (B and C) rings has no vinyl group and shows no peak near 988 cm-1. These observations verify the assignment of the 988 cm-1 peak of bilirubin to its pendant vinyl groups. This supports our previous proposal that a decrease in the peak at 985-995 cm-1 in the i.r. spectra of pigment gallstones, as compared with unconjugated bilirubin or calcium bilirubinate, indicates a consumption of vinyl groups in the process of formation of the polymer in the pigment stones.     

Atypical kinetic behavior of chloroperoxidase-mediated oxidative halogenation of polycyclic aromatic hydrocarbons

We have identified an atypical kinetic behavior for the oxidative halogenation of several polycyclic aromatic hydrocarbons (PAHs) by chloroperoxidase (CPO) from Caldariomyces fumago. This behavior resembles the capacity of some members of the P450 family to simultaneously recognize several substrate molecules at their active sites. Indeed, fluorometric studies showed that PAHs exist in solution as monomers and π-π dimers that interact to different extents with CPO. The dissociation constants of dimerization were evaluated for every single PAH by spectrofluorometry. Furthermore, docking studies also suggest that CPO might recognize either one or two substrate molecules in its active site. The atypical sigmoidal kinetic behavior of CPO in the oxidative halogenation of PAHs is explained in terms of different kinetic models for non-heteroatomic PAHs (naphthalene, anthracene and pyrene). The results suggest that the actual substrate for CPO in this study was the π-π dimer for all evaluated PAHs.     

An Antibody Specific for Component 8 of Myelin Basic Protein from Normal Brain Reacts Strongly with Component 8 from Multiple Sclerosis Brain

Myelin basic protein (MBP) consists of several components or charge isomers (C-1 through C-8) generated by one or a combination of posttranslational modifications. One of these, C-8, has been shown to contain citrulline (Cit) at defined sites formed by deimination of six arginyl residues. This unusual modification has allowed us to raise antibodies specific for this charge isomer only. To do this, a synthetic peptide, Gly-Cit-Cit-Cit-Cit, was coupled to keyhole limpet hemocyanin and injected into rabbits. The antibodies so generated reacted only with C-8 and not with any of the other charge isomers. A second antibody fraction was raised against the synthetic peptide ACitHGFLPCitHR naturally occurring between residues 24 and 33 of C-8 (all other charge isomers contain R instead of Cit at positions 25 and 31). These antibodies preferred C-8 but reacted with the other charge isomers, to the extent of approximately 25-30% of the reactivity shown with C-8. In studies with C-8 from multiple sclerosis (MS) MBP, much greater reactivity was obtained with these antibodies when compared with their reactivity with C-8 from normal MBP. Because the total number of Cit residues in C-8 from MS and normal MBP is the same, the difference in reactivity may be related to structural factors. The antibodies raised with the tetra-Cit peptide were reacted with three pairs of synthetic peptides: 24ARHGFLPRHR33 and ACitHGFLPCitHR; 120GQRPGFGYGGRAS132 and GQCitPGFGYGGCitAS; and 157GGRDSRSGSPMARR170 and GGCitDSRSGSPMACitR. They reacted only with the Cit-containing peptides in the order 157-170 greater than 120-130 greater than 24-33.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of oleic acid nitration by *NO(2)

Fatty acid nitration is a recently discovered process that generates biologically active nitro lipids; however, its mechanism has not been fully characterized. For example, some structural details such as vinyl and allyl isomers of the nitro fatty acids have not been established. To characterize lipids that originated from a biomimetic reaction of *NO(2) with oleic acid, we synthesized several isomers of nitro oleic acids and studied their chromatography and mass spectra by various techniques of mass spectrometry. LC/MS analysis performed on a high resolution micro column detected molecular carboxylic anions of various oleic acid nitro isomers (NO(2)OA). Esterification of NO(2)OA with pentafluorobenzyl bromide and diisopropylethylamine as a catalyst produced a unique isoxazole ester derivative exclusively from allyl NO(2)OA isomers via dehydration of the nitro group at ambient temperatures. This new analytical procedure revealed that *NO(2) generated two vinyl and two allyl isomers of NO(2)OA. The vinyl isomers showed high regioselectivity with the 1.8:1 preference for the 10-NO(2)OA isomer that was absent among allylic isomers. The nitration also generated elaidic acid via cis-trans isomerization and diatereoisomers of vicinal nitro hydroxy, nitro keto and alpha-nitro epoxy stearic acids with high stereo and regioselectivity. Nitration of small unilamelar phospholipid vesicles resulted in several phospholipids containing nitro lipids and elaidic acid amenable to hydrolysis by phospholipase A(2).     

Molecular modelling of the interactions of tetra-(4-N-methylpyridyl) porphin with TA and CG sites on DNA.

The molecular structure of the DNA-intercalating ligand tetra-(4-N-methylpyridyl) porphin has been determined by X-ray crystallography. The porphyrin has a precise centre of symmetry; the central core is planar, with the N-methylpyridyl groups inclined to it at angles of 66-72 degrees. Molecular modelling of this structure into TpA and CpG sites of intercalated DNA, has been performed, and approximate energetics calculated. It has been shown that only the CpG site can have full ligand intercalation, since the thymine methyl group sterically hinders such geometry at TpA sites. Modelling indicates the importance of electrostatic effects in the low-energy forms of intercalated and part-intercalated complexes at both sequences.     

New cytotoxic diterpenylnaphthohydroquinone derivatives obtained from a natural diterpenoid

Diterpenylquinone/hydroquinone derivatives were prepared through Diels-Alder cycloaddition between natural myrcecommunic acid or its methyl ester and p-benzoquinone (p-BQ), using BF(3).Et(2)O as catalyst or under microwave (Mw) irradiation. Acetyl, methyl and benzyl derivatives of several diterpenylnaphthohydroquinone were prepared from cycloadducts following two basic synthetic strategies, either protection before aromatisation or viceversa. Some of them were further functionalised at the B-ring of the decaline core. Most of the new compounds were evaluated and some of them resulted cytotoxic against several tumour cell lines with IC(50) values under the microM level.     

Donor specificity and regioselectivity in Lipolase mediated acylations of methyl α-d-glucopyranoside by vinyl esters of phenolic acids and their analogues

Methyl α-d-glucopyranoside as a model acceptor was acylated by several phenolic and non-phenolic vinyl esters using immobilised Lipolase. Donor specificity and regioselectivity of reaction were investigated. Conversion and rate of acylation by structurally varied donors indicates that the synthetic reactivity of Lipolase corresponds to the hydrolytic activity of feruloyl esterase type A. Lipolase exhibited remarkable regioselectivity for primary position of methyl α-d-glucopyranoside. The acylation occurred exclusively at 6-O primary position when vinyl esters of phenolic acids (hydroxybenzoates, hydroxyphenylalkanoates and hydroxycinnamates) served as acyl donors (5–77%). In addition to the major 6-O-acyl products (52–79%), 2,6-di-O-acylated derivatives were isolated from reaction mixtures (2–13%) when non-phenolic donors were used (vinyl esters of fully methoxylated derivatives of phenolic acids, along with vinyl benzoates, cinnamates or some heterocyclic analogues).     

Plasmonic DNA: Towards Genetic Diagnosis Chips

The present paper summarizes some of our activities in the field of plasmonic DNA and genetic diagnosis, presenting our system and its capabilities before showing data related to the design and use of functionalized biochips of increasing complexity along with various experimental hybridization conditions, including solutions containing one type of purified synthetic short oligonucleotides or PCR-amplified DNA samples from patients. The diagnosis capability of our system was evaluated by detecting several point mutations that alter the function of the CFTR gene and cause cystic fibrosis, a frequent monogenic disorder selected as a clinical model system.     

One-electron photooxidation of porphyrins at low temperature

The π-cation radicals of the metalloporphyrins magnesium octaethylporphyrin (MgOEP), magnesium tetraphenylporphyrin (MgTPP), and zinc tetraphenylporphyrin (ZnTPP), as well as the free base porphyrins of tetratolylporphyrin (H₂TTP) and tetraphenylporphyrin (H₂TPP) have been formed at liquid nitrogen temperatures in a rigid matrix of alkyl chloride glasses containing CCl₄ or 1,1,2,2-tetrachloroethane (TCE), following photolysis of the porphyrins with visible light. The reaction proceeds via electron transfer from the photoexcited porphyrin to the solvent molecules; the efficiency of thie electron transfer may be qualitatively evaluated in terms of electron tunneling in the solid matrices. This is the first report of the photochemical formation of a free base porphyrin π-cation radical species.     

Experiments Directed toward the Total Syntheses of Polycyclic Terpenes

A synthetic sequence for the conversion of 2-methoxycarbonyl-methyl-3-methoxy-carbonyl-4-methylindanone to (±)-7-deoxyepiallo-gibberic acid methyl ester norketone is described.

A synthetic sequence for conversion of m-methoxy benzaldehyde to 2-methoxycarbonyl-methyl-3-methoxycarbonyl-5-methoxy indanone and its reaction with methyl vinyl ketone are described.     

In situ synthesis of (5-phenyl-1H-pyrazole-3-carboxylic acid) metal complexes and their stable supramolecular microporous frameworks

By in situ generation of ppc ligand in the presence MCl₂ and 3-phenyl-1H-pyrazole-5-carbohydrazide at room temperature, the complexes, [M(ppc)₂(DMF)₂]·2H₂O (M = Co (1) and Ni (2); ppc = 5-phenyl-1H-pyrazole-3-carboxylic acid), were synthesized and characterized by single-crystal X-ray diffraction, IR spectrum, UV spectra and elemental analysis. X-ray structural analysis revealed that the complexes were formed into 3D supramolecular polymers via C-H?O and π?π interactions. The 3D supramolecular networks of two complexes were constructed from 1D nanochannels along the c-direction with the disorder DMF and water solvent molecules shuttling easily. TG and powder X-ray diffraction analysis showed two complexes had high stability when DMF and H₂O molecules were removed. Upon DMF molecules liberated, two complexes become stable microporous solid with coordination-unsaturated [M(ppc)₂] as center. The electrochemical properties were also investigated.     

Occurrence of fatty acid chlorohydrins in jellyfish lipids.

Fatty acid chlorohydrins are characterized as lipid components of an edible jellyfish. The four isomers 9-chloro-10-hydroxypalmitic acid, 10-chloro-9-hydroxypalmitic acid, 9-chloro-10-hydroxystearic acid, and 10-chloro-9-hydroxystearic acid were identified by gas chromatography-mass spectrometry comparison of the methyl esters and their trimethylsilyl derivatives with known synthetic samples. Two additional isomers, 11-chloro-12-hydroxystearic acid and 12-chloro-11-hydroxystearic acid, were also found in the lipid by the identification of the expected mass spectral fragments of the trimethylsilyl (Me3Si) derivative of their methyl esters. These six isomeric compounds represented approximately 1.4% of the total extractable jellyfish lipid and were released from the lipid as methyl esters by boron trifluoride-methanol treatment. These isomers account for only about 30% of the organic chlorine in the lipid. Evidence is given that the remaining organic chlorine is also present as fatty acid chlorohydrins containing more than one hydroxyl group.